Snail Expression In Ovarian Clear Cell Adenocarcinoma And 17 β Estradiol's Effect On Snail Expression

Objectives:To investigate the expression of ER, Snail and E-Cadherin in ovarian clear cell adenocarcinoma, Snail' effect on E-cadherin and MMP-2 expression, and 17βEstradiol' impact on Snail expression in ovarian clear cell adenocarcinoma. Materials and Methods:ERα, ERβ, MTA3, snail, E-cadherin and MMP-2 mRNA expression of ovarian clear cell adenocarcinoma cell line ES-2 and ovarian serous adenocarcinoma cell line SKOV3 were measured by RT-PCR, and snail protein expression was detected by IHC, and ERα, ERβ, E-cadherin protein exprssion were measured by Western Blot, and MMP-2 activity detected by Zymography. ER, snail and E-cadherin expression in paraffin embedded specimen of 20 patients of ovarian clear cell adenocarcinoma and 26 patients of ovarian serous adenocarcinoma were detected by IHC.Plasmid pRNAT-U6. 1/Neo-Snail containing short hairpin RNA of Snail was constructed to transfect ES-2 and SKOV3 cell line. Then cell adhesion was measured by adhesion assay, the invasion and mobility assay were performed by using the modified Boyden chambers, E-Cadherin and MMP-2 mRNA and protein expression were measured by methods mentioned above.ES-2 and SKOV3 cell line were grown in medium depleted of steroid for more than 7 days. Treated by 10^-7M, 10^-8M and 10^-9M 17βEstradiol, cell viability of ES-2 was determined by MTT method, and cell cycle distribution and apoptosis were detected by flow cytometry(FCM). The invasion and mobility assay was performed by using the modified Boyden chambers. MTA3, snail and MMP-2 mRNA expression of ES-2 and SKOV3 cell were measured by RT-PCR, and snail, MMP-2 protein expression was detected by IHC, and MMP-2 activity detected by Zymography. 17βEstradiol was added after Snail RNA interference, then change of MMP-2 mRNA expression and enzyme activity of ES-2 were detected. Results:Compared with SKOV3, ES-2 cell line was characteristic of negative ER a expression, low expression of MTA3, high expression of Snail, negative expression of E-cadherin and high expression of MMP-2. IHC results of paraffin embedded specimen showed that compared with ovarian serous adenocarcinoma, ovarian clear cell adenocarcinoma was characteristic of low ER and E-Cadherin expression, and high Snail expression. In ovarian clear cell adenocarcinoma, expression of Snail was not correlated with clinical phase, high Snail expression was observed even in patients of phase I . Expression of Snail was negatively correlated with that of ER and E-Cadherin, and expression of ER and E-Cadherin was positively correlated.When Snail expression was blocked by RNA interference, cell morphology of ES-2 and SKOV3 cell line was changed, cell adhesion was improved(P<0.05), and cell invasion and mobility was downregulated (/^O. 05). mRNA and protein expression of E-Cadherin was elevated, and mRNA and protein expression of MMP-2 was downregulated.17 P Estradiol 10⁷mol/L, 10⁸mol/L and 10^W/L all stimulated cell proliferation. 173 Estradiol 10"8mol/L reduced the proportion of G0-G1 phase(P <0.05), increased the proportion of S phase (P <0.05), but it had no effect on apoptosis. E 10"8mol/L significantly improved invasion and mobility of ES-2 cell (/><0.05). 10⁸mol/L 17 P Estradiol led to reduced mRNA expression of MTA3, and elevated mRNA and protein expression of Snail and MMP-2 in ES-2 and SKOV3 cells, and reduced mRNA and protein expression of E-cadherin in SKOV3 cells. When Snail expression with blocked by RNA interference, 17P Estradiol' s effect on cell invasion and upregulation of MMP-2 expression was blocked. Conclusions:Comparing with ovarian serous adenocarcinoma, ovarian clear cell adenocarcinoma was characteristic of low ER and E-Cadherin expression, and high Snail expression. Its high invasiveness could be attributed to its high Snail expression.In ES-2 and SKOV3 cell line, Snail repressed the expression of E-Cadherin and upregulated the expression of MMP-2, thus downregulated cell adhesion and improves cell invasiveness.